We propose a novel approach to create single dose, multivalent, biodefense vaccines, using bacteriophage T4 nanoparticle delivery platform. In the two highly successful U01 biodefense grants (17 publications and 5 patents), we have developed a T4-protective antigen (PA) anthrax vaccine that provided complete protection to rabbits and rhesus macaques against 100 LD50 aerosol challenge of B. anthracis Ames spores, and a T4-F1mut-V (mutated capsular antigen F1 and low calcium response V antigen) plague vaccine that provided complete protection to mice and Brown Norway rats against intranasal or aerosol challenge with 5,000 LD50 of Yersinia pestis CO92. Building on these successes, this proposal will achieve two major goals: i) develop a single dose anthrax-plague dual vaccine that can protect against both inhalation anthrax and pneumonic plague, and ii) establish a plug and play T4 vaccine delivery platform that can be adapted to any biodefense or emerging pathogen. The anthrax PA and plague F1mut-V antigens will be displayed on the 120nm x 86nm phage T4 capsid shell, as fusion proteins of the small outer capsid protein, Soc (870 copies). The genes corresponding to these antigens will be cloned under the control of a strong CMV promoter and packaged inside the capsid, using the phage T4 DNA packaging machine. The DNA molecules will induce production of antigens in the host for weeks to months. A series of nanoparticle formulations will be prepared containing PA and F1mut-V antigens, genes, or both (Aim 1). Mice will be immunized with a single dose of these formulations, without an adjuvant, and screened for protection against both anthrax and plague, using our newly developed anthrax- plague double challenged model. Of particular interest are the prime-boost vaccines containing antigen outside and DNA inside. The protein would act as prime and the DNA as boost, by continually expressing the antigens, and, in turn, stimulating potent immune responses. Immunological analyses will quantify binding antibody titers, lethal toxin neutralization titers, and cellular responses. The best vaccine formulations will be further evaluated for durability of the responses as well as the ability to induce mucosal immunity by oral delivery (Aim 2). The two best formulations from the mouse studies will be tested in a second animal model, New Zealand white rabbit for inhalation anthrax and Brown Norway rat for pneumonic plague (Aim 3). Finally, their immunogenicity and protective efficacy will be evaluated in the cynomolgus macaque model by double aerosol challenge with Y. pestis CO92 followed by Ames spores of B. anthracis (Aim 4). Synergized by the development of a robust vaccine delivery platform, state of the art facilities, partnership with an established industry partner, and a team of highly accomplished investigators with complementary strengths, this proposal would lead to the creation of an efficacious anthrax-plague dual vaccine that will have tremendous impact on the biodefense vaccine program.